An artificially split class 3 intein

Inteins excise themselves from precursor polypeptides through protein splicing, joining N- and C-exteins with a peptide bond. Split inteins are expressed as separate polypeptides that undergo protein trans splicing (PTS). Here, we demonstrate PTS can be achieved using an artificially split class 3 intein. Because class 3 inteins use an internal initiating nucleophile near the C-extein junction, rather than the first residue of the intein, both catalytic nucleophiles are present on a single polypeptide. This results in a compact arrangement of catalytic nucleophiles for PTS compared to the standard arrangement for split class 1 inteins.


Description
Intervening proteins (inteins) are translated within host proteins and excised via protein splicing (Paulus, 2000).In this reaction, the intein rearranges the two flanking peptide bonds into one, in the process ligating the adjacent N-and C-exteins and escaping the host protein (ligated exteins) without a trace.The ability of inteins to perform this chemistry has been exploited for a variety of protein engineering applications.
The class 1 mechanism of protein splicing occurs in four steps (Mills et al., 2014).First, an N-terminal intein residue (Cysteine or Serine) performs a nucleophilic attack on the peptide bond between the N-extein and intein, resulting in a (thio)ester linkage.Second, the first residue of the C-extein, a Cysteine, Serine, or Threonine, performs a second nucleophilic attack on the (thio)ester resulting from step 1, forming a branched intermediate.Third, the last residue of the intein, often a conserved asparagine, cyclizes to release the intein.Lastly, a peptide bond is formed between the N-and C-exteins through (thio)ester rearrangement.Class 2 and 3 splicing mechanisms have also been described.Class 3 inteins utilize a very similar strategy as class 1 inteins, except for the position of the initiating nucleophile that begins the splicing reaction -the initiating nucleophilic attack is carried out by an internal residue near the C-terminal intein-extein junction.
Inteins are particularly abundant in archaea and bacteria, but are also present in phages, viruses, and unicellular eukaryotes (Novikova et al., 2016).Inteins can be full-length, meaning the intein has an internal homing endonuclease domain (HEN), which assists in the invasion of intein-free alleles and makes some inteins mobile genetic elements.Mini-inteins lack the HEN domain, but retain conserved splicing blocks necessary for splicing.Interestingly, some inteins have been split in nature and are synthesized as separate polypeptides that undergo protein trans splicing (PTS).
Inteins can be thought of as self-contained, single-turnover enzymes with a unique ability to break and remake peptide bonds at designated positions.As such, this ability has been exploited in numerous protein engineering applications including bioseparations, bioconjugation, biosensing, and protein cyclization (Wood and Camarero, 2014;Sarmiento and Camarero, 2019).The impact of intein-based technologies on protein engineering has been, and continues to be, profound.Split inteins provide a unique avenue to control the protein splicing reaction by physical separation of intein fragments.Split inteins typically divide into larger N-intein (~100 residues) and smaller C-intein (~40 residues) domains (Eryilmaz et al., 2014;Aranko et al., 2014).Upon reassociation, split inteins adopt a highly similar fold to cis splicing inteins.
Numerous split inteins using a class 1 splicing mechanism have been extensively engineered to improve rate, accuracy, and controllability of PTS (Eryilmaz et al., 2014;Aranko et al., 2014), and only recently has there been the demonstration of trans splicing using alternative chemistries (Pinto et al., 2020;Hoffmann et al., 2021).In this work, we artificially split the class 3 DnaB1 intein from Mycobacterium smegmatis (DnaBi1) and observe efficient splicing in trans.The major difference between this system and traditional split intein systems is that both the initiating and +1 nucleophiles, as well as conserved terminal asparagine of the intein, are on the same Int C -C-extein fragment.The expected mechanism of class 3 trans splicing is shown (Figure 1A).
The replicative helicase DnaB from M. smegmatis houses two inteins (DnaBi1 and DnaBi2).DnaBi1 is a mini-intein (139residues) that utilizes a class 3 splicing mechanism, while DnaBi2 is a full-length intein that employs class 1 chemistry.The crystal structure of DnaBi1 shows a disordered loop between conserved splicing blocks, consistent with the expected Hedgehog/Intein (Hint) domain fold (Kelley et al., 2018).This disordered loop, located ~40 residues from the intein-C-extein junction, is where most naturally trans splicing inteins are split.Several class 1 cis splicing inteins have been divided at this loop, resulting in engineered trans splicing (Aranko et al., 2014).We sought to determine whether the class 3 M. smegmatis DnaBi1 could splice in trans.As the initiating nucleophile (C118) for M. smegmatis DnaBi1 is located after the disordered HEN loop, both catalytic nucleophiles would be housed on the C-intein-extein fragment.This design results in protein trans splicing with a compact arrangement of catalytic nucleophiles.
We introduced a split in DnaBi1 within the disordered HEN loop between residues 94 and 95 (Figure 1B) and measured trans splicing using a modified version of an established splicing reporter, MIG (Kelley et al., 2018).In the MIG reporter (MBP-Intein-GFP), the intein (surrounded by 10 native extein residues) is flanked by the non-native exteins MBP (Maltose Binding Protein; N-extein) and superfolder GFP (sfGFP; C-extein).sfGFP-containing products can be detected using in-gel 9/22/2023 -Open Access fluorescence following semi-native PAGE (Weinberger II and Lennon, 2021).MBP-DnaBi1 N and DnaBi1 C -sfGFP were coexpressed from different promoters on the same plasmid in E. coli and sfGFP-containing products were measured.Two products were observed, the smaller of which is consistent in size with DnaBi1 C -sfGFP (32.8 kDa) and the larger with the expected product from trans splicing between MBP-DnaBi1 N and DnaBi1 C -sfGFP, MBP-sfGFP (69.7 kDa) (Figure 1C).
Previously, it was demonstrated that the initiating nucleophile C118 of DnaBi1 was required for splicing in cis (Kelley et al., 2018;Woods et al., 2020;Lennon et al., 2021).We next tested whether this residue is also required for splicing in trans.As expected, and consistent with splicing between MBP-DnaBi1 N and DnaBi1 C -sfGFP, the larger product is completely absent when the C118A substitution is made on DnaBi1 C -sfGFP (Figure 1C).The requirement of both of C118 for splicing in cis and loss of the band consistent in size with MBP-sfGFP only when C118 is mutated strongly suggests protein trans splicing between MBP-DnaBi1 N and DnaBi1 C -sfGFP.It may be possible for the larger sfGFP-containing product to be a dimer of Int C -sfGFP (65.6 kDa), rather than MBP-sfGFP, as the initiating nucleophile C118 is free to form a disulfide.Additionally, while samples were not boiled to maintain sfGFP structure prior to semi-native SDS-PAGE, ß-mercaptoethanol was added to reduce any potential disulfide bonds and eliminate this possibility.
Finally, to further confirm that trans splicing had occurred, we reasoned that the larger sfGFP-containing band must also contain MBP.To determine whether the larger sfGFP-fluorescent product also contained MBP, we tested capture by amylose resin, which will pull-down any MBP-tagged products.Indeed, we found that the larger sfGFP-containing band was retained by the resin (Figure 1D).Taken together, these results clearly demonstrate the formation of MBP-sfGFP from coordinated MBP-DnaBi1 N and DnaBi1 C -sfGFP expression, a product that can only result from class 3 trans splicing.
Here, we show that a well-studied, small, conditionally-regulated (Kelley et al., 2018;Woods et al., 2020;Lennon et al., 2021) class 3 intein DnaBi1 from M. smegmatis can be artificially split for PTS applications.Due to the catalytic nucleophile arrangement of a class 3 intein, this design results in a compact arrangement of nucleophiles for trans splicing.As split inteins technologies based on class 1 inteins have been exceptionally useful in protein engineering, alternative intein chemistries may prove useful for certain applications.

Protein expression
Cells were grown in LB with 100 µg/mL ampicillin at 37ºC to mid-log (OD 600 ~0.5), and protein expression was induced by addition 1 mM Isopropyl β-d-1-thiogalactopyranoside (Gold Bio).Protein expression proceeded for either 20 hours at either 16ºC or 21ºC after which cells were pelleted by centrifugation.

Amylose resin purification
Following protein expression (MBP-DnaBi1 N or MBP-DnaBi1 STOP ), cells were resususpened in 20 mM Tris (pH 7.5), 200 mM NaCl (Amylose Column Buffer) with 1X HALT Protease Inhibitor Cocktail (Thermo Scientific), and lysed by sonication.Crude lysate was centrifuged at 20,000 x g, 4ºC and supernatant was applied to Amylose Resin (New England Biolabs) equilibrated in Amylose Column Buffer, washed with ~20 column volumes of Amylose Column Buffer, and eluted with Amylose Column Buffer plus 10 mM maltose.

SDS-PAGE
For in-gel fluorescence, cells were resuspended in 50 mM Tris-HCl (pH 8.0), 20 mM NaCl, 10% glycerol and lysed by sonication.Insoluble material was removed by centrifugation and soluble lysate was mixed with 4X Laemmli Sample Buffer with 4% ß-mercaptoethanol (Bio-Rad).Samples were separated using 8-16% Tris-Glycine TGX gels (Bio-Rad).Samples were not heated to maintain sfGFP structure and fluorescent products were detected with an Amersham Imager 680 (GE Healthcare).